A strain of transmissible gastroenteritis virus (TGEV), designated H16, was isolated in PK-15 cells and passaged serially to level 165. Vaccines based on passages 155-165 in cell cultures are available commercially as vaccines for the prevention and control of infections with TGEV in China. Nucleoprotein (N) sequences of the virus at passages 155 and 165 were aligned and compared using a computer software program. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiation of the vaccine strain from the other TGEVs was investigated. The RFLP analysis identified a change in the cleavage sites of AclI at passages 155 and 165. This RFLP pattern of the N gene differentiated the Chinese vaccine strain from its parental strain, the 11 TGEVs studied and the other reported TGEVs in the GenBank. Using phylogenetic analysis, the Chinese TGEVs were divided into three groups (G1, G2, and G3). The G3 Chinese TGEVs possessed several specific nucleotides and amino acids that were not found in the G1 and G2 Chinese TGEVs or the other reference TGEVs. Analysis of the phylogenetic trees revealed that the G3 TGEVs represent a separate group that is distinct from the non-Chinese TGEVs and from Chinese TGEVs isolated previously. These findings suggest that Chinese strains of TGEV are evolving continuously.